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| Home > Publications database > Untersuchungen zur Sekretion und zum Abbau von L-Threonin in Corynebacterium glutamicum |
| Home > Publications database > Untersuchungen zur Sekretion und zum Abbau von L-Threonin in Corynebacterium glutamicum |
| Dissertation / PhD Thesis/Book | PreJuSER-26992 |
2000
Forschungszentrum, Zenralbibliothek
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/20650
Report No.: Juel-3816
Abstract: The aerobic, Gram-positive soil bacterium Corynebacterium glutamicum is an effective producer of amino acids. It is able to excrete L-threonine which is economically important as an additive for animal feed. Recently, it was shown that an efficient production of L-threonine with C. glutamicum is limited by its intracellular degradation and by the low capacity of an assumed L-threonine export carrier. Hence, the present work describes the investigation of L-threonine degrading enzymes in C. glutamicum as well as the identification and characterization of the L-threonine export carrier gene . Enzymatic investigations revealed that L-threonine is converted to glycine and acetaldehyde. This aldol cleavage was shown to be catalyzed by serine hydroxymethyltransferase (SHMT). The corresponding gene glyA was isolated and sequenced. However, since glyA was proved to be essential for C. glutamicum its inactivation was not possible . Therefore, a strain with a single chromosomal copy of glyA under control of the IPTG inducible tac-promoter was constructed. In this strain SHMT activity could be down-regulated by low IPTG concentrations to 10% of the enzyme activity of the wild type. Previously, biochemical analyses have revealed that L-threonine efflux in C. glutamicum is carrier-mediated . To identify and clone the corresponding export carrier gene, an appropriate screening system for export deficient mutants was established. It was shown that the addition of the tripeptide Thr-Thr-Thr to the medium led to retarded growth due to intracellular accumulation of L-threonine. Export deficient mutants should be unable to grow under these conditions due to extremely high intracellular amounts of L-threonine. A transposon mutant bank of C. glutamicum was constructed. By using the tripeptide screening system, nine mutants were isolated that exhibited retarded growth in presence of Thr-Thr-Thr. Analysis of the insertion sites of the transposon showed that in one of these mutants the inactivated gene was the L-threonine export carrier gene thrE. This gene encodes a hydrophobic membrane protein which does not show homology to any known transporter. It is 489 amino acids in size and is predicted to possess nine putative transmembrane helices. It was proved that L-threonine export is correlated with thrE expression . Inactivation of thrE resulted in a reduced export rate for L-threonine of 1 .0 nmol min" mg"' dry weight, compared to 2 .5 nmol min" mg" dry weight for the wild type, whereas with overexpressed thrE L-threonine was exported at a rate of 3.8 nmol min"' mg"' dry weight . Furthermore, the substrate specificity of the L-threonine export carrier was investigated . In addition to L-threonine also L-serine is transported by ThrE. Overexpression of thrE in combination with reduced L-threonine degradation led to an increase of extracellular accumulated L-threonine of about 50% in a L-threonine producing strain of C. glutamicum.
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